The present invention relates to methods for measuring oxidation parameters of low density lipoproteins (LDL), which methods are rapid, simple to perform, and valid for the determination of LDL oxidation products and LDL antioxidant potential. These methods provide specific means for assessing the oxidative stress in the body of an individual in general and, in particular, for assessing or screening the risk for, and for the diagnosis, management and research of atherosclerosis and coronary heart disease.
Oxidation of low-density lipoprotein plays a key role in processes leading to the development of atherosclerosis. LDL oxidation is accompanied by alterations in its biological properties resulting in, for example, accelerated uptake through scavenger receptors in macrophages, altered chemotactic behavior of monocytes, and monocyte-derived macrophages, endothelial cell damage, and increased amounts of mediators of cell proliferation and platelet aggregation (refs. 1-4). All these effects may contribute to the development of atherosclerotic lesions. Therefore, determination of the LDL oxidation related parameters, namely LDL oxidation products, and antioxidant potential, gives more specific information on atherosclerosis-related biochemical phenomena than the commonly used measurements, of which the most common are the measurement of serum cholesterol, LDL and other lipoproteins and the apolipoproteins.
Most of the data on LDL oxidation come from studies where oxidation of LDL fractions, isolated by conventional ultracentrifugation methods, has been monitored by the appearance of conjugated dienes or thiobarbituric acid reactants arising during oxidation of isolated LDL in vitro (5). Thus far, when LDL oxidation has been investigated in humans in vivo, analyses of LDL oxidation products have been based on antibodies raised against in vitro oxidatively damaged LDL (5). The existing methodology is complex and time-consuming and, in addition, the specificity of the immunological analyses can be questioned (3). Therefore, there is still need for single rapid and specific measurement of LDL oxidation that could become part of the laboratory repertoire in the diagnosis and management of atherosclerosis (5).
The immunological methods developed for direct measurement of oxidized LDL may not be specific, as, in addition to oxidized LDL, antibodies seem to recognize also other epitopes (6) and have given contradictory results as well (3). The poor applicability of immunological methods may be a reflection of the chemistry of LDL oxidation: LDL oxidation can be initiated in various different polyunsar fatty acids, and each of these can give rise to a number of different kinds of oxidation products. Due to the multiplicity of oxidation products, development and use of immunological methods is likely to remain problematic also in the future.
The existing methods for measuring the antioxidant potential of LDL are complex and time consuming, and for example only a limited number of analyses can be performed within one working week: LDL is first isolated by ultracentrifugation, whereafter the samples still have to be dialyzed. Another disadvantage is the unprecise recording of results, where changes of the various reaction phases are not always easily detected.
We have developed, for the analysis of LDL oxidation parameters, namely LDL oxidation products and LDL antioxidant potential, methods which are rapid and simple to perform, and can therefore be used for large-scale clinical studies. The validity and clinical applicability of these analytical procedures is clearly indicated by several studies.
The objects of the present invention are fulfilled by providing a kit for use in the screening of the risk for, the diagnosis, management and research of atherosclerosis and coronary heart disease comprising means for isolating LDL from a serum or plasma sample for the preparation of a LDL fraction, and means for separating the lipids from the LDL fraction to obtain a lipid fraction.
In a preferred embodiment of this invention, the means for isolating the LDL from the serum or plasma sample is a buffered heparin solution and the means for separating the lipids is a chloroform-methanol solution.
According to a further embodiment, the kit comprises a means for use in the determination of the baseline level of conjugated dienes (LDL-BDC) in the lipid fraction. Said means is preferably an organic solvent, and more preferable cyclohexane.
It is a further object of this invention to provide a kit for use in the screening of the risk for, the diagnosis, management and research of atherosclerosis and coronary heart disease comprising means for isolating LDL from a serum or plasma sample for the preparation of a LDL fraction, and means for use in the determination of the antioxidant potential of LDL (LDL-TRAP) in the sample.
According to a preferred embodiment, the means for isolating the LDL from the sample is a buffered heparin solution, the means for use in the determination of the antioxidant potential of LDL in a serum or plasma sample is 2,2xe2x80x2-azobis(2-amidinopropane)HCl (ABAP). The LDL-TRAP is preferably determined by using chemiluminescence.
It is still a further object of this invention to provide a kit for use in the screening of the risk for, the diagnosis, management and research of atherosclerosis and coronary heart disease comprising means for isolating LDL from a serum or plasma sample for the preparation of a LDL fraction, means for separating the lipids from the LDL fraction to obtain a lipid fraction, means for use in the determination of LDL-BDC in the lipid fraction, and means for use in the determination of the antioxidant potential of LDL (LDL-TRAP) in the sample.
Further areas of applicability of the present invention will be apparent from the detailed description given hereinafter.
The kits of the present invention can comprise a combination of the individual components needed to screen the risk for, diagnose, manage and research atherosclerosis and coronary heart disease presented together in a common pack. For this purpose, the kit can comprise separate vials or containers for the necessary reagents and substrates.
According to another aspect of the invention, improved kits and assay methods are provided. The kits are useful for quantifying oxidation parameters in a LDL fraction, optionally in a pre-isolated LDL fraction. The kits include in separate containers reagents for determining baseline levels of conjugated dienes, as described herein, in improved ratios and amounts. Other kits are usefull for quantifying antioxidant potential in a LDL fraction of blood serum or plasma, optionally in a pre-isolated LDL fraction. Such kits contain in separate containers unexpectedly improved and novel formulations, ratios and quantities of reagents for determining total peroxyl radical trapping antioxidant potential. The reagents include precipitants for precipitating an LDL fraction from blood or serum, solvents and solvent mixtures for extracting lipids from the LDL fraction, resuspension solvents and solvent mixtures for resuspending the LDL fractions and/or lipids isolated therefrom, and detection reagents for enabling or improving the detection of measurable assay end products. The improvements in methodology and equivalent instructions with the kits of the invention include sample handling, mixing and centrifugation parameters as are described herein.
Thus in some embodiments, kits for use in quantifying oxidation parameters of lipids in a LDL fraction of blood serum or plasma are provided. The kits include a first container for extracting the lipids from the LDL fraction, the first container containing a solvent which extracts lipids from a LDL fraction and a second container containing an amount of resuspension solvent sufficient to resuspend the extracted lipids. In certain embodiments, the solvent which extracts lipids is chloroform:methanol having a ratio greater than about 2:1, preferably greater than about 3:1, and more preferably about 4:1. In other embodiments, the resuspension solvent in the second container is neutral or inert to spectrophotometric analysis. Preferably the resuspension solvent in the second container is cyclohexane. Instructions for measuring absorbance at 234 nm and 300 nm, as well as calculation of LDL-BDC from the absorbance also can be included.
In still other embodiments, kits for use in determining antioxidant potential of a LDL fraction of blood serum or plasma are provided. The kits include a first container for extracting lipids from the LDL fraction, the first container containing a solvent which extracts lipids from a LDL fraction, and a second container containing an amount of a compound which produces peroxyl radicals sufficient to induces lipid peroxidation of the LDL fraction. Preferably the compound in the second container is 2,2xe2x80x2-azobis(2-amidinopropane)HCl (ABAP). In some embodiments, the ABAP is in a dry or solid form such as a powder and the kit includes a third container containing a solution for suspension of the ABAP. In other preferred embodiments, the kits include a third container containing a compound which enhances luminescence, which preferably is luminol. The kits also can include instructions for use of greater than about 25 xcexcg of luminol per assay, more preferably greater than about 50 xcexcg luminol per assay, even more preferably greater than about 75 xcexcg per assay and most preferably about 88.5 xcexcg per assay.
Kits for isolation of LDL from a blood or serum sample are provided in other embodiments. The kits include a container containing a buffered heparin solution, and instructions for adding a sufficient quantity of the buffered heparin solution to the blood or serum sample to form a LDL precipitate, mixing the mixture gently, and centrifuging the resulting mixture for at least 20 minutes to recover the LDL precipitate.
Kits combining two or more aspects of the foregoing kits also are provided, including kits for measuring LDL-BDC and LDL-TRAP, etc.
The method for determining LDL-BDC is based on the determination of the xe2x80x9cdiene conjugation,xe2x80x9d by which is meant the rearrangement of the double bonds in polyunsaturated fatty acids, which is an early event in the process of lipid peroxidation (7). This rearrangement is known to cause a specific change in UV-absorption of the fatty acid molecule. This is advantageous in two important ways: (i) diene conjugation occurs only during peroxidation of polyunsaturated fatty acids; (ii) diene conjugation is a common step in peroxidation of all polyunsaturated fatty acid molecules. In experimental in vitro studies on LDL oxidizability, the diene conjugation is regarded as the most reliable index, and is widely used in this context (5). The LDL-BDC method, however, is the first method used for the direct measurement of LDL diene conjugation in vivo without oxidation of the serum or plasma sample.
The method for measuring the antioxidant potential (LDL-TRAP) is, in contrast to the previously known methods, rapid (isolation of LDL and the measurements are done within the same day) and accurate.